Purification of -aminobutyric acid (GABA) receptor and formation of monoclonal antibody against the purified receptor.

γ-氨基丁酸（GABA）受体的纯化和针对纯化受体的单克隆抗体的形成。

• 批准号: 59480120
• 负责人: KURIYAMA Kinya
• 金额: $ 4.48万
• 依托单位: Kyoto Prefectural University of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1984
• 资助国家: 日本
• 起止时间: 1984 至 1986
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-59480120/
• 关键词: GABA receptor; Benzodiazepine Receptor; C1 Channel; Functional Coupling; Functional Reconstitution; Receptor Antibody; Immunohistochemistry; Distribution in Brain; Glycoprotein; クロライドチャンネル; 機能的共役

Purification of solubilized -aminobutyric acid (GABA) and benzodiazepine (BZP) receptors from the bovine cerebral cortex was exployes by the affinity columu chromatography using a benzo-diazepine, 1012-s, as immobilized ligand. The gel filtration using Sephadex G-200 of the pufified GABA/BZP receptor complex revealed the molecular weight of the receptor complex was approximately 340,000. SDS-polyacrylamide gel electrophoretic profiles of the 1012-S-eluted fraction showed the existence of two major bands having the molecular weights of approximately 53,000 and 57,000, both of which were irreversibly photoaffinity-labeled with [^3H]flunitrazepam. The purified gaba receptor was found to be a glycoprotein and its carbohydrate chain of a hybrid type was involved in the functional coupling between GABA receptor and BZP receptor. The GABA receptor antibody formed by biweekly subcutaneous injection of the purified GABA receptor to Albino rabbits has revealed immunohistochemically that this receptor complex in the rat brain is localized various brain areas such as the somata of Purkinje cells and gramule cells layer of the cerebellum, large cells in the pars compacta of substantia nigra, pyramidal cells in the cerebral cortex (layeers III-V) and hippocampus, respectively.

用亲和柱层析法，以苯二氮卓类化合物1012-S为固定化配基，从牛大脑皮层中分离纯化可溶性γ-氨基丁酸（GABA）和苯二氮卓类化合物（BZP）受体。用Sephadex G-200对纯化的GABA/BZP受体复合物进行凝胶过滤，结果表明受体复合物的分子量约为340，000。1012-S洗脱组分的SDS-聚丙烯酰胺凝胶电泳图谱显示存在两条主要条带，分子量分别约为53，000和57，000，这两条条带均用[^3H]氟硝西泮进行了不可逆的光亲和标记。纯化的GABA受体是一种糖蛋白，其杂合型糖链参与GABA受体与BZP受体的功能偶联。将纯化的GABA受体两周一次皮下注射给白化病兔，形成GABA受体抗体，化学分析表明，这种受体复合物在大鼠脑内分布于不同的脑区，如小脑的浦肯野细胞和颗粒细胞层的胞体，黑质腹侧部的大细胞，大脑皮质（III-V层）和海马的锥体细胞。

项目成果

K.Kuriyama: "Psychotropic and Neurotropic Drugs and Neurotransmitter Receptors" Excerpta Medica, 87 (1987)

K.Kuriyama：“精神药物和神经药物以及神经递质受体”医学摘录，87 (1987)

K. Kuriyama,;S. Tomono,;M. Kishi,;T. Mukainaka,;S. Ohkuma: "Development of GABAergic neurons in cerebral cortical neurons in primary culture." Brain Research. 416. 7-21 (1987)

K.栗山，；S。

K.Kuriyama;J.Taguchi: Journal of Neurochemistry. 48. 1897-1903 (1987)

K.Kuriyama；J.Taguchi：神经化学杂志。

J. Taguchi; K. Kuriyama: "Functional coupling of -aminobutyric acid (GABA) and benzodiazepine type II receptors: Analysis using purified GABA/benzodiazepine receptor complex from bovine cerebral cortex." Neuropharmacology. 26. 1945-1950 (1987)

J.田口；

K. Kuriyama: J. Taguchi: "Glycoprotein as a constituent of purified GABA/benzodiazepine receptor complex: Structures and Physiological roles of its carbohydrate chain." Journal of Neurochemistry. 48. 1897-1903 (1987)

K. Kuriyama：J. Taguchi：“糖蛋白作为纯化 GABA/苯二氮卓受体复合物的组成部分：其碳水化合物链的结构和生理作用。”